AtxA is the master regulator of Bacillus anthracis virulence and is critical for disease in many animal models for anthrax. AtxA was first characterized as a positive regulator of anthrax toxin gene expression, but the protein also affects transcription of the capsule biosynthetic operon and many other genes, resulting in far- reaching effects on the overall physiology of the bacterium. Despite the importance of AtxA, the molecular basis for its function and mechanisms controlling its activity are largely unknown. Our recently solved crystal structure of AtxA, supported by our biochemical studies, places the protein within a class of transcriptional regulators with activities known or predicted to be controlled by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) of Gram-positive bacteria. In the current model for PTS-mediated control of these regulators, one or more histidine residues in PTS-regulatory domains (PRDs) are phosphorylated by PTS proteins in response to the presence or absence of an inducing sugar. AtxA contains two domains closely matching established PRDs. As expected for PRD-containing regulators, AtxA activity is affected by phosphorylation at specific histidine residues in the PRDs. Nevertheless, our studies of AtxA have revealed key differences from the prevailing model for PTS control. PRD-containing regulators have been investigated primarily in non-pathogens, but in recent years discoveries of PRD-containing virulence gene regulators (PCVRs) in a growing list of pathogens have suggested relationships between carbohydrate signaling/metabolism and expression/function of critical virulence factors. The AtxA structure, combined with our biochemical and genetic studies, in vivo system for quantifying AtxA activity, and murine model for anthrax make us well-positioned to address mechanisms that control activity and expression of AtxA and paralogous PCVRs of B. anthracis. We will: (1) Investigate the mechanisms for Hpr control of atxA transcription. We hypothesize that independent of CcpA, Hpr(H14~P) controls transcription of atxA by one of two mechanisms: i) phosphorylation of a downstream regulator or ii) direct interaction with a protein other than CcpA; (2) Assess the role of phosphorylation in control of AtxA activity. We hypothesize that phosphorylation of AtxA by a protein other than Hpr alters the affinity and/or specificity of AtxA DNA-binding domains for DNA sequences in promoter regions of target genes; and (3) Determine the target specificity of AtxA and its paralogues, AcpA and AcpB and relationships to specific PCVR domains. We hypothesize that (i) the B. anthracis PCVRs have common and specific target genes, and the pleiotropic effects of AtxA on transcription are mediated in part by the downstream regulators AcpA and AcpB, and (ii) unique PRDs of the PCVRs determine target specificity. Investigations proposed here will increase our specific knowledge of Bacillus anthracis virulence gene control, while having a broader impact on our understanding of a central metabolic regulatory system that is common among Gram-positive bacteria and linked to virulence in many pathogens.